Cysteine or thiol proteases contain a reactive sulphydral moiety activated by an adjacent histidine. Hydrolysis of the substrates peptide bond is initiated when the cysteine sulfur attacks the carbon in the peptide bond forming a thiol-enzyme intermediate, liberating the amino portion of the peptide. The thiol-enzyme intermediate is hydrolyzed by water releasing the substrates C-terminus and restoring the enzyme. There are over 20 some families of cysteine proteases. [Rawlings N. D., & Barrett A. J. Families of cysteine peptidases. Methods in Enzymol. 244 461–486 (1994)]. The present invention relates to a thiol protease of the C2 family that includes the calpain superfamily.
Calpains are calcium-activated intracellular neutral cysteine proteases (EC 3.4.22.17)(for reviews see Sorimachi et al., Structure and physiological function of calpains. Biochem J. 328:721–32, 1997; Carafoli E and Molinari M. Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193–203, 1998). Some calpains are expressed ubiquitously while others are tissue-specific. μ-Calpain and m-calpains appear in all tissues, p94 is skeletal muscle specific while nCL-2 is stomach specific. (Sorimachi et al., Structure and physiological function of calpains. Biochem J. 328:721–32, 1997). The best characterized are μ-calpain and m-calpains which consist of two subunits. An 80 kDa large subunit contains both Ca2+ binding sites and the catalytic activity and small 30 kDa subunit with a separate set of Ca2+ binding sites. All the proteolytic activity is contained in the larger subunit of both μ- and m-calpain. In the presence of PEG or chaperones the large subunit is catalytically activated in the absence of the smaller subunit. Other calpains, for example nCL-2 and p94, are proteolytically active monomers with homology to the μ-calpain and m-calpains large subunit.
The large (catalytic) subunit has four domains (I–IV)(Hosfield et al., Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J. 18:6880–9, 1999; Strobl et al., The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci USA. 97:588–92, 2000). The N-terminus (domain I) contains an alpha helical region and a site of autocatalytic cleavage. Domain II contains the catalytically active domain with the active site amino acids (m-calpain residues Cys105, His262, & Asn286). Domain III contains the linker between the Ca2+ binding domain (in domain IV) and links Ca2+ binding to proteolytic activity. Domain IV contains a calmodulin-like Ca2+ binding regions with EF hands. p94 (also called calpain 3) is similarly organized with domains I–IV, but, also contains a proline-rich N-terminus and two unique insertion loops (IS1 and IS2). nCL-2 is also active as a large monomer with domains I–IV; however, a splice variant (nCL-2′) lacks domains III & IV, but maintains proteolytic activity.
Calpains are responsible for limited intracellular proteolytic cleavage, as opposed to complete proteolytic digestion. The proteolysis modifies protein function both specifically and irreversibly. Numerous proteins have been identified as calpain substrates (Carafoli E and Molinari M Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193–203, 1998; Hayes et al., Drug News Perspect 11:215–222, 1998). The best-characterized substrates are large cytostructural and/or membrane associated proteins, calmodulin-binding proteins and transcriptional factors. Physiologically significant substrates for calpain include kinases, phosphatases, channel proteins and cytoskeletal proteins that link transmembrane receptors to the membrane skeleton. Proteolytic modification of these proteins may have fundamental roles in development, differentiation, and cellular transformation in response to cell signaling, cell-cell and/or cell-extracellular matrix interactions. In platelets, calpain activation appears to be linked to clustering of the integrin receptor aIIb3 (Fox JE On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb Haemost 82:385–91, 1999).
Calpains have been implicated in cell signaling through activation of protein kinases and phosphatases (cleaving between regulatory and catalytic domains resulting in changes in activity after hydrolysis) and modulation of their intracellular localization. Calpains have been shown to modify specific enzymes and cytoskeletal proteins as part of calcium-mediated signal pathways. They are also involved in remodeling and disassembling the cytoskeleton, especially where the cytoskeleton attaches to membranes or other subcellular structures.
Several nuclear transcription factors have been suggested as calpain substrates. Calpains are also involved in the progression of cells through the cell cycle (Carafoli E and Molinari M Calpain: a protease in search of a function? Biochem Biophys Res Commun 247:193–203, 1998) in that calpain activity accelerates some cells through the cell cycle by cleavage of p53. Calpain is also thought to play a role in long term potentiation (memory) and rat strains deficient in the endogenous calpain inhibitor, calpastatin, have increased long term potentiation.
Calpains in Disease:
Several diseases have been associated with calpain deficiencies. For example, limb-girdle muscular dystrophy (LGMD) is a group of disorders that primarily cause weakness of the shoulder and pelvic regions. A subtype of LGMD called LGMD2A is caused by defects in the gene for p94 (also called calpain 3)(Richard et al., Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81:27–40, 1995).
Positional cloning has recently identified single-nucleotide polymorphisms (SNPs) in an intron of the gene coding for calpain-10 that appears to confer insulin resistance in diabetics. Presence of this mutation correlates with reduced levels of calpain 10 in patients susceptible to type II diabetes (Horikawa et al., Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet. 26:163–75, 2000). The same calpain-10 SNP also correlates with type II diabetes in a high-risk population of Pima Indians (Baier et al., A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest. 106:R69–73, 2000).
Over Activation of Calpain—Ischemic and Traumatic Damage
Intracellular calcium levels and calpain activity are normally tightly regulated. Under stress, such as follows neuronal excitotoxicity, ischemic stroke, hemoragic stroke, hypoxic stress and/or trauma, intracellular calcium levels rise causing inappropriate calpain proteolytic activity. Calpain activity has been implicated in further cell destruction and non-specific calpain inhibitors have been shown to be protective in animal models (Lee et al., Proc. Natl. Acad. Sci. USA, 88:7233–7237, 1991; Wang K K and Yuen P W. Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. 15:412–9, 1994; Lee, K S, et al., Calcium-activated proteolysis as a therapeutic target in cerebrovascular disease. Annal NY Acad Sci. 825, 95–103, 1997).
Calpains are activated in neurons following ischemia-induced damage in animal models of stroke. (Lee et al., Proc. Natl. Acad. Sci. USA, 88:7233–7237, 1991). Inhibition of calcium-activated proteolysis by means of high doses of (usually non-specific) calpain inhibitors protect against the degeneration of vulnerable hippocampal neurons after ischemia (Rami et al., Brain Research, 609:67–70, 1993; Wang et al., An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective. Proc Natl Acad Sci USA. 93:6687–92, 1996). After an ischemic insult, neuronal death is delayed for hours to days. This time interval represents a potential therapeutic window in which to apply effective therapies to minimize brain damage after stroke.
In addition to neuronal damage, calpains are thought to contribute to cardiac ischemic damage (Iwamoto H et al., Calpain inhibitor-1 reduces infarct size and DNA fragmentation of myocardium in ischemic/reperfused rat heart.J Cardiovasc Pharmacol 33:580–6, 1999) and hepatocyte necrosis during and following anoxia (Arora A S et al., Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation. J Cell Physiol 167:434–42, 1996).
Neurodegenerative Diseases
Calpains have been implicated in neurodegenerative diseases ncluding, Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease and amyotrophy. Calpain activation is increased during normal aging and a strong case can be made for the involvement of calpain in the abnormal proteolysis underlying the accumulation of plaque and neurofibriles in brain tissue from people who suffered Alzheimer-type dementia (Iwamoto et al., Brain Research, 561:177–180 1991; Nixon et al., Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease. Ann NY Acad Sci ;747:77–91, 1994; Grynspan et al., Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease. Brain Res 763:145–58, 1997). Calpains are significantly activated in human postmortem brain from patients with Alzheimer's disease, and the degree of activation correlated with those regions of the brain showing the greatest amount of degeneration (Saito et al., Proc. Natl. Acad. Sci. USA, 90:2628–2632, 1993). More recently, it has been recognized that in Alzheimer's disease cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are involved in neurite outgrowth and cortical lamination. Calpain cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimers disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5 which hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons (Lee et al., Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature. 18;405:360–4, 2000). Compounds that inhibit calpain activity could prove useful in reducing or delaying neurodegeneration caused to Alzheimer's disease.
Damage Following Trauma
Traumatic injury also causes calpain activation associated with further cell death, atrophy and shrinkage of the brain. A forceful blow trigger cell damage and increased calpain activity that can cleave structural proteins in the brain for up to weeks afterward (Hayes et al., Potential Contribution of Proteases to Neuronal Damage Drug News & Perspectives 11, 1998).
Calpain activation has also been implicated in spinal cord injury following trauma (for reviews see: Banik et al., Role of calpain and its inhibitors in tissue degeneration and neuroprotection in spinal cord injury. Ann. N.Y. Acad. Sci. 825:120–7 1997; Banik et al., Role of calpain in spinal cord injury: effects of calpain and free radical inhibitors. Ann. N.Y. Acad. Sci. 844:131–7, 1998). Analogous to brain trauma, secondary pathophysiological alterations occur in the traumatized spinal cord well after the initiating insult. These secondary events ultimately cause cell death and tissue damage. Non-specific calpain inhibitors have shown utility in preventing further damage due to spinal chord injury in animal models (Ray et al., Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection. Neurochem Res. 25:1191–8, 2000).
These studies indicate the potential utility of calpain inhibitors (especially those calpains located in the spinal cord) in treating traumatic injury resulting from automobile crashes, gunshot wounds, and sports accidents.
Degeneration of Cochlear Tissues Following Noise Exposure
Calpains are activated during acoustic trauma and calpain inhibitors protect against hearing loss caused by noise (Stracher A Calpain inhibitors as therapeutic agents in nerve and muscle degeneration. Ann NY Acad Sci 884:52–9, 1999).
Inflammation
Calpains also regulate integrin-mediated interaction of T-cells with the extracellular matrix (ECM) and calpain inhibitors prevent acute and chronic inflammation in animal models (Cuzzocrea S et al., Calpain inhibitor I reduces the development of acute and chronic inflammation Am J Pathol 157:2065–79, 2000).
Multiple Sclerosis
Multiple sclerosis is characterized by the progressive loss of the myelin of the brain and spinal cord. In autoimmune demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis, the degradation of myelin proteins results in the destabilization of the myelin sheath. Calpains have been implicated in that calpain degrades all major myelin proteins and increased calpain activity is observed in multiple sclerosis (Shields D C et al., A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain. Proc. Natl. Acad. Sci. USA 96:11486–91, 1999).
Cataract Formation
In the lens, crystallins prevent thermal denaturation and aggregation of other proteins. Crystallins are also substrates for calpains and cataract formation in a rat model of selenite-induced cataract formation is thought to result from calpain activation and cleavage of crystallins (Shearer T R, David L L, Anderson R S, Azuma M. Review of selenite cataract. Curr Eye Res 1992; 11:357–369). In this model the crystallin cleavage could be blocked by calpain inhibitors (Azuma M et al., Cysteine protease inhibitor E64 reduces the rate of formation of selenite cataract in the whole animal. Curr Eye Res 10:657–666, 1991). In a genetic model cataract-prone rats also showed enhanced proteolysis of crystallins and lens cytoskeletin proteins thought to be mediated by calpain (Inomata M et al., Evidence for the involvement of calpain in cataractogenesis in Shumiya cataract rat (SCR). Biochim Biophys Acta 1362:11–23 1997). Calpain activation is also thought to play a role in cataracts induced by buthionine sulfoximine, calcium ionophore A23187, hydrogen peroxide, diamide, xylose, galactose and streptozotocin (Kadoya et al., Role of calpain in hydrogen peroxide cataract. Curr Eye Res 1993; 12:341–346; David et al., Buthionine sulfoximine induced cataracts in mice contain insolubilized crystallins with calpain II cleavage sites, Exp Eye Res 1994; 59:501–504.). These models of cataract formation in rats suggest that calpain-induced proteolysis is a common underlying mechanism. Fragments of alpha-crystallin, consistent with calpain cleavage, have been also observed in cataractous human lens.
Reovirus Induced Myocarditis
Infection of neonatal mice with reovirus produces histological myocarditis. This is due to a direct viral injury and apoptosis of myocytes. Calpain inhibitors block reovirus-induced apoptosis in vitro and prevented viral-induced induced myocarditis (DeBiasi et al., Calpain inhibition protects against virus-induced apoptotic myocardial injury. Virol 75:351–61, 2001).
The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length novel CAN-12 calpain gene, its encoded polypeptide, in addition to the variants CAN-12v1 and CAN-12v2. Also provided are polypeptide alignments illustrating the strong conservation of the CAN-12, CAN-12v1, and CAN-12v2 polypeptides to known proteases and a model of the active conformation of CAN-12. Based on this strong conservation, the inventors have ascribed the CAN-12, CAN-12v1, and CAN-12v2 polypeptides as having calpain proteolytic activity. Data is also provided illustrating the unique tissue expression profile of the CAN-12 polypeptide in esophagus, lymph node, and spinal cord tissues.
In fact, calpains have been the subject of significant research and development programs designed to identify inhibitors of this disease associated protein class. For example, the following, non-limiting examples of drugs, therapies, or regimens directed to inhibiting calpains are currently known: BDA 410 (Mitsubishi Tokyo); AK 295 (Alkermes; CAS® Registry Number: 160399-35-9, 144231-82-3, and 145731-49-3; (1-(((1-ethyl-3-((3-(4-morpholinyl)propyl)amino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid phenylmethyl ester stereois); AK 275 (Alkermes; CAS® Registry Number: 158798-83-5, and 150519-08-7; N-((phenylmethoxy)carbonyl)-L-leucyl-N-ethyl-L-2-aminobutanamide); inhibitor 1 (University of Indiana; acetyl-leu-leu-norleucinal); calpeptin (University of Indiana; benzyloxycarbonyl-leu-norleucinal); VASOLEX (Cortex); RESTENEX (Cortex); MDL 28170 (Aventis; CBZ-Val-Phe-H); PI (Sankyo; CAS® Registry Number: 128102-74-9, and 128102-75-0; L-phenylalanyl-L-glutaminyl-L-valyl-L-valyl-3-((3-nitro-2-pyridinyl)dithio)-L-alanylglycinamide); MDL 28170 (Hoechst Marion Roussel); BDA-410 (Mitsubishi-Tokyo); SJA-6017 (Senju; CAS® Registry Number: 190274-53-4; Butanamide,2-(((4-fluorophenyl)sulfonyl)amino)-N-((1S)-1-formyl-3-methylbutyl.).-3-methyl-, (2S)-); Pharmaprojects No. 5123 (Pfizer; 2-Chloro-acetic acid(3-oxo-4-phenyl-3,4-dihydro-1H-quinoxalin-2-ylidene)hydrazide; WO96-25403); CEP-4143 (Cephalon; WO96-14067); MDL-104903 (Aventis; CAS® Registry Number: 180799-56-8; Carbamic acid, ((1S)-1-(((4S,5R)-5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl)-2-methylpropyl)-, phenylmethyl ester)); MDL-28170 (Aventis; CAS® Registry Number: 19542-51-9; Alanine, N-(N-carboxy-L-valyl)-3-phenyl-N-benzyl ester, L-); CX-275 (Cortex Pharmaceuticals; PhenylmethylN-((1R)-1-((((1S)-1-ethyl-3-(ethylamino)-2,3-dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamate); NS 7 (Nippon Shinyaku; 4-(4-Fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride); Calpain inhibitor 1 (Suntory; N-Acetyl-L-leucinyl-L-leucinyl-L-norleucinal); E64 (Taisho Pharmaceutical (; CAS® Registry Number: 66701-25-5); CEP 4143 (Cephalon); SJA 6017 (Senju; N-(4-Fluorophenylsulfonyl)-L-valyl-L-leucinal); The present invention is directed to antagonists specific to the CAN-12, CAN-12v1, and/or CAN-12v2 polypeptides. Modulating the activity of the calpain polypeptides of the present invention may result in fewer toxicities than the drugs, therapies, or regimens presently known to regulate other known calpains.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of CAN-12, CAN-12v1, and CAN-12v2 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the CAN-12, CAN-12v1, and CAN-12v2 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides, particularly activators and inhibitors of the novel CAN-12, CAN-12v1, and CAN-12v2 polypeptides of the present invention.